Next Article in Journal
Application of Telepathology for Rapid On-Site Evaluation of Touch Imprint Cytology in CT-Guided Percutaneous Transthoracic Core Needle Biopsy of Pulmonary Nodules: The Experience of Our Multidisciplinary Thoracic Tumor Board
Previous Article in Journal
Changes of Prostate-Specific Membrane Antigen-Radioligand Uptake on PET with Systemic Therapy in Patients with Metastatic Renal Cell Carcinoma
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Cancers
Volume 17
Issue 11
10.3390/cancers17111737
cancers-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Synthesis and Preliminary Cytotoxicity Evaluation of 3-Lup-20(29)-Ene-3β,28-Diol Glycoconjugates Containing a Succinic Linker and a 1,2,3-Triazole Ring

by
Julia Szreder
1,
Klaudia Woźniak
1,
Karol Erfurt
2,
Mirosława Grymel
1,3,* and
Gabriela Pastuch-Gawołek
1,3,*
1
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, B. Krzywoustego 4, 44-100 Gliwice, Poland
2
Department of Chemical Organic Technology and Petrochemistry, Silesian University of Technology, B. Krzywoustego 4, 44-100 Gliwice, Poland
3
Biotechnology Center, Silesian University of Technology, B. Krzywoustego 8, 44-100 Gliwice, Poland
*
Authors to whom correspondence should be addressed.
Cancers 2025, 17(11), 1737; https://doi.org/10.3390/cancers17111737
Submission received: 2 April 2025 / Revised: 9 May 2025 / Accepted: 21 May 2025 / Published: 22 May 2025
(This article belongs to the Section Cancer Drug Development)
Browse Figures
Versions Notes

Simple Summary

3-Lup-20(29)-ene-3β,28-diol (betulin, BN) is a natural bioactive compound with broad biological activities, especially anticancer, antibacterial, anti-inflammatory, and antiretroviral. However, poor bioavailability and low intracellular accumulation limit its pharmaceutical application. A promising strategy to enhance BN’s therapeutic potential is glycoconjugation. We developed an efficient method for modifying the betulin backbone at position C28 with sugar units via a (CO)CH2CH2COOH linker, based on CuAAC, yielding ten new betulin glycoconjugates with good yields and purity confirmed by spectroscopic analysis (NMR, HRMS).

Abstract

Background: 3-Lup-20(29)-ene-3β,28-diol (betulin, BN) is a natural bioactive compound with significant synthetic and pharmacological potential. A growing body of research highlights the increasing interest in BN and its derivatives, driven by their broad biological activities (anticancer, antibacterial, anti-inflammatory, antiretroviral). However, poor bioavailability and low intracellular accumulation limit its pharmaceutical application. Methods: A promising strategy to enhance BN’s therapeutic potential is glycoconjugation. This approach improves drug bioavailability, solubility, and selectivity, particularly in cancer therapy, by leveraging cancer cells’ heightened glucose demand and overexpression of glucose transporters. Incorporating an N-heterocyclic linker, such as a 1,2,3-triazole ring, further enhances biological activity. Results: We developed an efficient method for modifying the betulin backbone at position C28 with sugar units via a (CO)CH2CH2COOH linker, based on CuAAC, yielding ten new betulin glycoconjugates with good yields and purity confirmed by spectroscopic analysis (NMR, HRMS). The potential for inhibition of cancer cell proliferation (HCT-116 human colorectal carcinoma cell line and MCF-7 human breast cancer cell line) and cytotoxicity toward normal human dermal fibroblasts (NHDF-Neo) was assessed. Conclusions: The obtained glycoconjugates exhibited higher activity against MCF-7, indicating the selectivity of their action. The development of glycoconjugates based on increased glucose demand and overexpression of its transporters could be an interesting strategy for acquiring anticancer agents, combining innovative chemical solutions with biological complexity. Such an approach may be crucial in the effective fight against cancer diseases.

1. Introduction

3-Lup-20(29)-ene-3β,28-diol, commonly known as betulin (BN), is an example of a natural bioactive substance with significant synthetic and pharmacological potential (Figure 1). Scientific articles and patents available in various databases highlight researchers’ growing interest in betulin. Its abundant availability, low cost, and structurally versatile parent framework make betulin an excellent candidate for the design of new derivatives with improved pharmacokinetic properties, particularly enhanced bioavailability [1,2,3]. Despite its wide range of biological activity, betulin has limitations, such as poor solubility and bioavailability. The parent skeleton therefore requires structural modifications to release its full therapeutic potential.
Therefore, research in this area is justified, allowing for the expansion of the library of BN derivatives, and enhancing the information on the structure-biological activity correlation.
One promising approach to improving the properties of bioactive substances is glycoconjugation, which involves attaching sugar units to the biologically active backbone. The sugar moiety can be linked to the active compound by forming a glycosidic bond or using various types of linkers. The linker may also contain structural elements important from the point of view of biological activity [4]. This strategy offers several advantages, including the ability to improve water solubility and bioavailability while improving selectivity toward specific molecular targets. Sugar moieties play a crucial role in directing glycoconjugates of biologically active compounds toward specific receptors or transporters that are overexpressed in cancer cells. This targeted delivery mechanism reduces off-target effects and increases the therapeutic efficacy of the compound [5]. The phenomenon that allows us to understand the role of sugars in targeted drug delivery in anticancer therapy is the Warburg effect. It explains that cancer cells have a different metabolism than healthy cells. Even with sufficient oxygen supply, anaerobic respiration associated with lactic acid fermentation dominates, as a result of which cancer cells convert glucose mainly into lactate [6,7]. The effect of this different metabolism is an increased demand for glucose in cancer cells and its enhanced transport across the cell membrane, which results in GLUT transporters overexpression [8,9,10]. The Warburg effect is particularly visible in the cases of colorectal [9,11], breast [12], lung cancers [13], or glioblastoma [14].
The glycoconjugation strategy has been used to modify the properties of anticancer drugs such as ifosfamide [15], gemcitabine [16], platinum derivatives [17], doxorubicin [18], or paclitaxel [19].
In the structure of many pharmaceuticals, both those already approved for use and those still under evaluation, an N-heterocyclic fragment is often present, and its presence has a significant impact on the biological activity [20,21]. Derivatives containing 1,2,3-triazole ring are of particular interest in drug design [22]. Such compounds have been widely studied for their antibacterial, antiviral, anti-inflammatory, anticancer, and antimicrobial activity [23]. Examples of drugs with a 1,2,3-triazole system in their structure include tazobactam, a β-lactam antibiotic [24], and savolitinib, a receptor tyrosine kinase (c-Met) inhibitor, approved in China for the treatment of selected types of cancer [25].
Based on this observation, it seems advantageous to connect the sugar with the BN backbone via a linker containing an N-heterocyclic system. This is possible due to the use of 1,3-dipolar azido-alkyne cycloaddition catalyzed by copper (CuAAC), which allows for a simple and efficient connection of the sugar and the bioactive compound via the 1,2,3-triazole ring [23].
A variety of triazole derivatives of betulin, including glycoconjugates, have been described in the scientific literature and evaluated for their biological activities. In vitro studies have revealed that some of the obtained compounds show promising antiviral, anticancer (cytotoxic and antiproliferative), and antimicrobial (antibacterial and antifungal) properties, with a specific activity profile depending on the substituents introduced and the compound’s structure. Structural modifications of the BN backbone using click chemistry reactions (CuAAC) can target the positions C3, C28, and C30, as illustrated in Figure 2.
In our previous study, we performed modifications of the betulin backbone, involving the introduction of a sugar unit via a 1,2,3-triazole ring at C3 and/or C28 positions of BN (mono- or disubstituted glycoconjugates, Figure 3). Unfortunately, cytotoxicity assay results of these compounds turned out to be unsatisfactory. Furthermore, 3,28-O,O’-bis(2-(4-(hydroxymethyl-1H-1,2,3-triazol-1-yl)acetyl)betulin, modified only with a triazole system, demonstrated unexpectedly high in vitro activity against the MCF-7 cell line. However, it also showed toxicity against healthy cells, which is a serious limitation to its therapeutic potential. This observation confirmed our belief that the presence of the sugar moiety is necessary to improve selectivity [28].
In this work, we proposed new BN glycoconjugates (Figure 4), considering that an important element is the linker structure. Under standard physiological conditions, such a linker should exhibit sufficient stability and controlled lability, to allow the glycoconjugate to reach its target without premature degradation. Therefore, as a stable and biocompatible linker element between pharmacophores, we chose a triazole system, which presents hydrolysis resistance and adequate stability in biological systems (amide bond mimetism). In addition, the presence of a triazole ring may improve the solubility of glycoconjugates in water, which is important for their bioavailability and potential for in vivo administration.
The solution to the problem of structure stiffening could be the introduction of a fragment derived from succinic acid into the structure of betulin glycoconjugates. It should not only make the entire molecule more flexible, but also strengthen the chelating properties of metal ions by the designed glycoconjugates [33]. It is noteworthy that cancer cell growth and their metastasis are strongly related to copper ions uptake [34]. Therefore, it seems that metal chelating agents may be a promising strategy for controlling copper levels in the body, and consequently may be one of the components of anticancer therapy [35,36,37]. An additional advantage of such a solution is the combination of the betulin skeleton and the sugar ligand with the linker by an ester bond, which should undergo hydrolysis after reaching the target site, thus allowing the release of the drug in the cell.

2. Materials and Methods

2.1. General Experimental Informations

All compounds were obtained according to general principles of organic synthesis. The crude products were purified by column chromatography, and the structure and purity of the obtained compounds were confirmed by spectroscopic analysis using NMR (nuclear magnetic resonance), HRMS (high-resolution mass spectrometry), and determination of the values of optical rotations. All chemical reagents used in the synthesis were purchased from ACROS Organics (Geel, Belgium) or Avantor (Gliwice, Poland) and were not additionally purified or dried before use in the synthesis. Synthesis descriptions, spectra, and their analysis are included in the Supplementary Materials (Schemes S1–S3).

2.2. Biological Studies

For biological studies three different cell lines were used: the NHDF-Neo cell line (Lonza, Poland), the HCT-116 cell line (American Type Culture Collection, ATCC, Manassas, VA, USA), and the MCF-7 cell line (purchased by the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland and gifted for our research). Cells were cultured in 96-well plates (Sarstedt, Nümbrecht, Germany) using RPMI 1640 or DMEM/F12 media (100 µL per well) supplemented with fetal bovine serum (FBS, EURx, Gdańsk, Poland), and with Antibiotic–Antimycotic Solution (Avantor, Gliwice, Poland). Cells were seeded and incubated for 24 h prior to treatment: HCT-116 cells at a density of either 1 × 104 or 7.5 × 103 cells per well depending on incubation time, NHDF-Neo cells at 1 × 104 per well, and MCF-7 at 5 × 103 cells per well.
Stock solutions of the tested compounds were prepared in DMSO and stored under appropriate conditions. Immediately prior to the experiment, these solutions were diluted to the desired concentrations using a growth medium. In no dilution was the DMSO content higher than 1% v/v. The resulting solutions were then used to treat cells (100 µL per well). The studies were carried out in the concentration range from 25 µM to 200 µM of the tested compound, with incubation times varying from 24 to 72 h, depending on the specific cell type. In parallel, the influence of copper ions on cellular proliferation was investigated. In this case, the procedure was analogous to the previously described one, with the difference that glycoconjugates were added in the culture medium with the addition of CuCl2. The final concentration of CuCl2 was 20 µM in each well. Cytotoxicity studies were also carried out on an example of an anticancer drug, doxorubicin, against all tested cell lines. For determination of cell viability, the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; ACROS Organics) was applied following the manufacturer’s protocol. Absorbance of the formazan, formed during the test and dissolved in 100 µL of DMSO, was measured spectrophotometrically at 570 nm using a plate reader (Epoch, Bio-Tek, Winooski, VT, USA).
The measured absorbance was analyzed to assess proliferation, where tested samples were compared with the untreated controls (100%). The results from MTT were presented as the mean of three experiments (biological replicates). For each experimental condition within a replicate, four technical repetitions were performed. Technical repetitions were averaged to obtain a single value per replicate. Statistical analyses were conducted using means of the biological replicates. The percentage of viable cells and the standard deviation (SD) were calculated in Excel (Microsoft Office 365, A3 for faculty), using the STDEV.S formula, which determines the standard deviation for a sample set. The IC50 values were determined by generating dose-response curves using GraphPad Prism software version 10.2.3. The results are expressed as the average value ± standard deviation (SD). To evaluate statistical significance, two-way analysis of variance (ANOVA) with replication was performed to assess the effects of compound type and concentration on cell proliferation. When significant main effects were observed (p < 0.05), pairwise comparisons between betulin (BN) and each analogue at corresponding concentrations were conducted using two-tailed unpaired t-tests. Bonferroni correction was applied to account for multiple comparisons, and adjusted p-values < 0.05 were considered statistically significant. All statistical tests were performed in Excel.

2.3. In Silico Analysis

Molecular descriptors (logP, TPSA, molecular weight, hydrogen bond acceptors and hydrogen bond donors) of BN glycoconjugates were estimated using the Molinspiration Cheminformatics 2025 platform (http://www.molinspiration.com, accessed on 8 February 2025). SMILES (Simplified Molecular Input Line Entry System) codes were generated using ChemBioDraw Ultra version 12.0 (Cambridge Software) based on structural formulas.

3. Results and Discussion

The concept of click chemistry involves using two building blocks: one containing a terminal alkyne group and the other featuring an azide functional group. Depending on the selection of these reagents results in obtaining conjugates with different spatial orientations of the 1,2,3-triazole ring in the linker structure (Scheme 1).
The results of our previous studies have indicated that carrying out the CuAAC reaction using a propargyl derivative of betulin and a sugar derivative containing an azide group is favorable, from the glycoconjugates cytotoxicity point of view [28]. In this work, the attention was focused on betulin glycoconjugates containing only one sugar residue attached at the C28 position of betulin. It was also decided to assess whether the presence of an acetyl group at the C3 position of the betulin skeleton affects the pharmacokinetic properties of the new glycoconjugates.
For this reason, 3-O-acetyl-28-O’-(3-propynylcarbonyloxypropanoyl)betulin 3a functionalized at the C28 position with succinic linker terminated by propargyl fragment, and its equivalent 3b with a free hydroxyl group at the C3 position, were obtained (Scheme 2).

3.1. Betulin Analogues with Alkyne Moiety (3)

In the first step of the synthesis, according to the protocol described by Thibeault et al. [38] with some modifications, 3,28-O,O’-diacetylbetulin and 3-O-acetylbetulin were obtained. Then, following the published procedure [39], 3-O-acetylbetulin was esterified at 125 °C for 18 h, with an excess of succinic anhydride (SA) in dry pyridine (Py), and in the presence of the catalytic amount of 4-(dimethylamino)pyridine (DMAP). The crude product was purified with chloroform extraction, followed by washing with water and HCl solution, and further column chromatography. 3-O-Acetyl-28-O’-(3-carboxypropanoyl)betulin 2a was obtained with a high yield (85%). The synthesis of 28-O-(3-carboxypropanoyl)betulin 2b was performed similarly, but reagents were used in a molar ratio of BN:SA (1:1.5), and reaction time was reduced to 8 h. Because of the higher reactivity of the primary hydroxyl group at the C28 position of the parent BN backbone (C28-OH) compared to the reactivity of tertiary OH group at the C3 position (C3-OH), the reaction time needed to be carefully controlled. It was possible to obtain product 2b with a high yield (67%).
The next step of our research involved the introduction of an alkyne moiety to the linker terminated by the carboxyl group ((O(CO)CH2CH2COOH). BN analogues (2a, 2b), dissolved in dimethylformamide (DMF) were reacted with propargyl bromide in the presence of potassium carbonate, in a molar ratio (2/HC≡CCH2Br/K2CO3, 1:6.6:3.5). After 5 days of stirring at room temperature, the product was isolated by extraction with diethyl ether, with 3a: 96%, 3b: 75% yields, respectively.

3.2. Sugar Derivatives (4)

Synthesis of sugar units was performed according to the previously described procedures [40,41,42,43,44,45] (the details are provided in the Supplementary Materials). The ability of GLUT to bind and transport glycoconjugates may be influenced by several factors, such as the carbohydrate structure, the position of its substitution and the linker type [46,47,48]. Due to that, for betulin glycoconjugation, both D-glucose and D-galactose derivatives (4a–4h) were selected, in which the hydroxyl groups were protected by acetyl or benzyl groups. The choice of protecting groups was dictated primarily by their sensitivity to enzymatic hydrolysis. Acetyl groups are relatively susceptible to the action of hydrolytic enzymes in the body. In turn, the benzyl groups exhibit greater stability and resistance to enzymatic hydrolysis, compared to acetyl groups. This makes them appear to be particularly useful in the context of transport across cell membranes.
According to literature reports, highlighting the importance of the substituent orientation at the anomeric position of sugars for effective binding to GLUT1 transporters [49,50,51], we designed and synthesized sugar derivatives 4a–4g with the β-configuration. The analysis of the crystal structure of human GLUT1 indicates that the hydroxyl group of D-glucose at the C6 position exhibits minimal involvement in the formation of hydrogen bonds with amino acid residues within this protein transporter [52]. Therefore, modification of the sugar unit at the 6-OH position should not affect receptor binding as strongly as modification at the anomeric position of the sugar. To maintain the affinity of the molecule to the GLUT1 transporter, it was decided to use a sugar 4h functionalized in the 6-OH position for glycoconjugation and link it through this position to the betulin derivative, i.e., the fragment responsible for inducing the cytotoxic effect. The structures of all sugar derivatives used for glycoconjugation of betulin derivatives are presented in Figure 5.

3.3. Synthesis of Betulin Glycoconjugates via Click Chemistry (5–8)

In the course of our research, we designed a library of new BN glycoconjugates, not described in the scientific literature so far, and then developed a method for their synthesis, using the concept of click chemistry as shown in Scheme 2. The copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition reaction (CuAAC) provides a simple and efficient method for the synthesis of molecular hybrids based on natural products, and its main advantage is the mild reaction conditions.
We have used building blocks, enabling the preparation of a whole range of glycoconjugates, containing in the structure a linker built from a succinyl fragment and a heteroaromatic 1,2,3-triazole ring, and in the case of compounds (7a–7d), an additional chain O(CH2)n. Such an elaborate linker was used to eliminate stiffening of the entire hybrid, which should ensure a better alignment of the molecule to sugar transporters.
The general procedure for glycoconjugation involves contacting of 3-O-acetyl-28-O’-(propynylcarbonyloxypropanoyl)betulin 3a or 28-O’-(propynylcarbonyloxypropanoyl) betulin 3b with the selected sugar unit (4a–4h) in the presence of copper(II) sulfate (CuSO4-5H2O) as a catalyst and sodium ascorbate (NaAsc) as a Cu(II) to Cu(I) reducing agent. The reactions were carried out in the solvent system water-isopropyl alcohol (i-PrOH)- tetrahydrofuran (THF) under an argon atmosphere at room temperature for 7 days. The reaction progress was monitored with TLC method. As a result, nine new glycoconjugates of BN (5–7) were isolated by column chromatography in very high yields, within the range of 53–73% (Table 1). Such a good yield was not achieved only in the case of glycoconjugate 8a (12% yield), which was attributed to the lower purity of the sugar derivative 4h used in the reaction. Unfortunately, when the unprotected galactose derivative 4e was used for the reaction, it was not possible to obtain the desired glycoconjugate. This was probably due to too low concentration of the sugar substrate in the reaction mixture, which is related to its poor solubility in the solvent system applied.

3.4. Synthesis of Metabolite (9a)

The molecules of betulin glycoconjugates, synthesized by our team, are built from several basic building blocks: (i) the betulin backbone, having a succinic linker at the C28 position, (ii) the sugar unit, and (iii) the linker, containing an aromatic 1,2,3-triazole ring. Inside the cell, the glycoconjugates can be subjected to the action of hydrolytic enzymes, resulting in the release of a potential metabolite. Given the stability of the triazole system under typical physiological conditions, we set out to synthesize and then assay the cytotoxicity of a potential metabolite 9a that could be formed under the action of hydrolytic enzymes.
In this case, we also carried out the CuAAC reaction, in which we used 3-O-acetyl-28-O’-(propynylcarbonyloxypropanoyl)betulin 3a and 2-azidoethanol in the presence of copper(II) sulfate (CuSO4-5H2O) and sodium ascorbate (NaAsc), in a molar ratio (3a/HC≡CCH2N3/ CuSO4-5H2O/ NaAsc, 1:1:0.2:0.4), in THF/iPrOH/water solvent system. We isolated metabolite 9a by column chromatography with 32% yield.
The structures of all synthesized compounds were confirmed by nuclear magnetic resonance (1H-, 13C-NMR) and high-resolution mass spectrometry (HRMS). NMR spectra of all newly synthesized products are presented in the Supplementary Materials.

3.5. Evaluation of Cytotoxicity

The series of structurally diverse betulin glycoconjugates underwent preliminary cytotoxicity screening using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide) [53]. Two cancer cell lines were chosen for the study: HCT 116 (colorectal carcinoma cell line) and MCF-7 (human breast adenocarcinoma cell line). This selection was not random, as in both types of cancer cell lines overexpression of glucose and galactose transporters is observed [54,55,56]. To determine if glycoconjugation enhanced the selectivity of the betulin derivatives, cytotoxicity tests on Normal Human Dermal Fibroblast-Neonatal cells (NHDF-Neo) were also conducted. The selection of this healthy cell line, so atypical in relation to the cancer cells tested, was based on the desire to compare the selectivity index determined for the currently obtained glycoconjugates with the results obtained for the betulin derivatives we described earlier [28] and draw preliminary conclusions on structure-selectivity relationships. All studies were carried out in the concentration range from 25 to 200 µM. The incubation time for individual cell lines varied from 24 to 72 h, due to differences in the doubling time of these cell lines. In the case of HCT 116 and NHDF-Neo cell lines, a shorter incubation time (24 or 48 h) was used, since, according to various literature sources, the doubling time for these cells ranges from 18 to 24 h [57]. In contrast, a longer incubation time (72 h) was chosen for MCF-7 cells, as literature reports on this cell line indicate a much longer doubling time of 35 to 72 h, depending on the source [58,59].
In the first step, the cytotoxicity of the building blocks, i.e., sugar units and betulin was assessed against the tested cell lines. While the sugar derivatives used for glycoconjugation proved to be inactive, betulin inhibited the proliferation of both cancerous and healthy cells. In this case, the difference in the determined IC50 values was negligible (34.88 ± 4.26 µM for HCT-116, 17.89 ± 1.06 µM for MCF-7, and 34.90 ± 8.36 µM for NHDF-Neo), indicating the need for modifications aimed to enhance the selectivity of betulin towards cancer cells.
In designing the molecular hybridmade of betulin and sugar unit, we focused on the differences in properties between cancerous and normal cells. The incorporation of a sugar moiety in the structure of the new hybrid was aimed at targeting the cells that overexpress sugar transporters, which is associated with the previously mentioned Warburg effect. Additionally, we considered factors that could enhance the affinity of betulin glycoconjugates for the interphase space. The presence of the basic 1,2,3-triazole ring should increase the concentration of glycoconjugates in the interphase space of cancer cells, which is known to have a mildly acidic environment, resulting from the cells’ excretion of lactate produced by excessive glycolysis in tumors [60,61]. The insertion of a 1,2,3-triazole ring together with a succinic linker was also intended to enable the formation of complexes with copper ions, which should further inhibit the proliferation of cancer cells by targeting a critical factor in their growth. Although patients with various malignancies, including colorectal and breast cancer, typically exhibit elevated levels of copper in their serum or tissues, cancer cells cultured in vitro are characterized by low copper levels [62,63]. Consequently, it seemed justified to conduct additional experiments with the addition of Cu2+ ions. First, the effect of a 20 µM copper chloride solution on the proliferation of cancer and healthy cells was assessed. Once confirmed that such a concentration of Cu2+ ions did not adversely affect the viability of the cell lines examined, we proceeded with further experiments aimed at investigating the influence of metal ion complexation by glycoconjugates on cell proliferation.
Figure 6 and Figure 7 illustrate the relationship between cell proliferation and concentrations of BN, glycoconjugates (5–8) or metabolite 9a. The IC50 values, defined as 50% cell growth inhibition compared to the untreated control, have been collected in Table 2.
The cytotoxicity of designed BN glycoconjugates against HCT 116 and MCF-7 is generally lower than that of native betulin. The tested glycoconjugates inhibited cell growth in a dose-dependent manner. After 24 h of treatment with the tested compounds, the HCT 116 cell line exhibited high survivability. After increasing the incubation time to 48 h, the HCT 116 cell line became more sensitive to the presence of the tested derivatives, particularly for glycoconjugates (7a–7d, Figure 6). For compound 7d, the IC50 value was determined to be 131.16 ± 8.76 µM. The results obtained indicate that for the HCT 116 cell line, the strategy of elongating the linker between the sugar moiety and the BN backbone was effective in reducing the stiffness of such a large molecule. Additionally, the presence of an unprotected hydroxyl group at the C3 position of BN proved to be significant for the biological activity of the glycoconjugates. Notably, the compounds tested displayed minimal cytotoxicity towards healthy NHDF-Neo cells (Figure 7), with the IC50 values remaining indeterminable for all conjugates within the tested concentration range.
Conversely, the MCF-7 cell line demonstrated the highest sensitivity to both BN and its glycoconjugates. Although for glycoconjugates the determined IC50 values were considerably higher than those for BN alone, it was possible to define the IC50 values for all tested conjugates (Table 2). One of the possible explanations for this state of affairs may be the proven and described in the literature overexpression of GLUT transporters (especially GLUT1) in this type of cancer [64]. However, confirmation of this thesis requires further studies using equivalents of the described glycoconjugates with a deprotected sugar moiety.
The most active glycoconjugates were (5c, 6a, and 7c), with determined IC50 values of 128.07 ± 1.35 µM, 122.61 ± 6.84 µM, and 134.92 ± 7.92 µM, respectively. Analyzing the relationship between the structure and cytotoxicity of the tested compounds, it is evident that the presence of an unprotected hydroxyl group at the C3 position of BN (as in the case of compounds 5c and 7c) plays a significant role. Notably, substituting the D-glucose residue with D-galactose significantly increased the cytotoxicity of compound 6a compared to the analogous derivative 5a that contains a D-glucose residue (IC50 values of 122.61 ± 6.84 µM and 163.12 ± 7.65 µM, respectively). Conversely, in this context, the rigidity of the linker between the sugar moiety and the BN derivative appeared to be less significant. The most active compounds did not include the two- or three-carbon alkyl chain typical of structures (7a–7d).
Interesting results emerged from an additional experiment designed to demonstrate the potential mechanism of action of the synthesized glycoconjugates, which involves the chelation of copper ions, essential for cancer cells growth. The antiproliferative activity of BN derivatives were evaluated in the presence of Cu2+ ions. To assess the impact of these complexes with metal ions on cell proliferation, a copper chloride solution was added to the growth medium, and the rate of cell growth in the presence of BN conjugates was compared to that of untreated cells. In the case of the HCT 116 cell line, a notable effect was observed for compounds (7a–7d), which had previously been identified as the most effective against these cells. After introducing copper ions, the IC50 values for compounds 7a and 7b were determined to be 150.08 ± 8.01 µM and 112.48 ± 0.57 µM, respectively. Remarkably, compound 7d exhibited the highest cytotoxic activity, with its IC50 value decreasing more than twofold under these conditions, compared to the value obtained in the absence of copper ions (60.40 ± 6.95 µM versus 131.16 ± 8.76 µM). For the NHDF-Neo cell line, only minor variations in cell viability were observed after treatment with the tested compounds alongside copper ions (Figure 7). This indicates that the viability of healthy cells was not as significantly affected by copper ion concentration as in the cancer cell line.
This observation highlights an increased selectivity of glycoconjugate 7d, compared to BN, as evidenced by the selectivity index (SI), calculated as the ratio of the IC50 value for healthy cells (NHDF-Neo) to the IC50 value for cancer cells. For BN, in the presence of copper ions, the SI value against HCT 116 cells was 1.6, whereas for glycoconjugate 7d, the SI value exceeded 3.3. In the case of the MCF-7 cell line, the introduction of copper ions also increased the cytotoxicity of the tested compounds, as indicated by the IC50 values. Notably, the most significant differences were observed for glycoconjugates 5c, 6a, and 8a, which exhibited IC50 values of 70.71 ± 5.44 µM, 59.81 ± 1.85 µM, and 88.33 ± 3.31 µM, respectively. For the most active glycoconjugate, 6a, the selectivity index (SI) improved to a value greater than 3.3, compared to a SI of 2.4 for BN. Interestingly, glycoconjugate 8a, which features the D-galactose derivative linked to BN via the C6 position, was not more effective than analogous glycoconjugate 6a, which is linked to BN through the C1 position (with IC50 values of 88.33 ± 3.31 µM and 59.81 ± 1.85 µM, respectively). This suggests that glycoconjugation through the C6 position in the sugar does not enhance the affinity of the final glycoconjugate for sugar transporters.
In the course of the studies, the cytotoxicity of potential metabolite 9a, which may be generated through the hydrolytic action of glycosidases on glycoconjugate 7a, was assessed. The relatively low cytotoxicity of this compound suggests that premature hydrolysis of glycoconjugates before reaching the target cancer cells is unlikely to pose a threat to healthy cells.

3.6. Molecular Descriptors

Determining the physicochemical properties of compounds is crucial in drug design, because these properties directly affect pharmacokinetics. One of the most widely known and used guidelines for assessing potential bioavailability is Lipinski’s Rule of Five (Ro5), proposed by Christopher Lipinski in 1997. This rule helps predict whether a compound can be effectively absorbed into the human body. According to Ro5, regarding orally administered drugs, a drug should meet the following criteria: molecular weight (MW) ≤ 500 Da, as larger molecules may have difficulty crossing cell membranes, thus affecting bioavailability. Additionally, the octanol–water partition coefficient (logP) should be ≤ 5; higher values indicate poor water solubility, which leads to lower bioavailability. The number of hydrogen bond donors should be ≤ 5, as groups like OH and NH can impact solubility, and an excessive number may hinder membrane permeability. The number of hydrogen bond acceptors should be ≤ 10, because its higher value can negatively affect drug absorption. Although not an absolute requirement, compounds that violate more than one of these criteria are less likely to be successful as oral drugs [65].
Another important descriptor to consider is the topological polar surface area (TPSA), which defines the molecule’s ability to penetrate biological membranes. Good oral absorption is typically associated with TPSA ≤ 140 Å2. However, certain compound classes do not fully follow these rules, but still demonstrate good pharmacokinetics. This is particularly true for antibiotics, biological drugs, and natural medical products [66]. It should be taken into account that the increasing diversity of drug compounds and therapeutic applications may require a more comprehensive strategy. Depending on the pharmacological class of the compound and its target profile, its bioavailability, solubility, and permeability may be affected [67].
Analyzing molecular descriptors plays a key role in the early stages of drug design. Using computational tools such as Molinspiration online software (http://www.molinspiration.com), scientists can predict the properties of a molecule and make structural modifications to improve bioavailability and efficacy, and also minimize potential problems related to toxicity or poor solubility. It is important to note, however, that there are exceptions to these rules.
Glycoconjugates (5a–c, 6a–b, 7a–d, and 8a, Table 3) have relatively high molecular weights exceeding 500 g/mol, and their TPSA also exceeds the 140 Å2 threshold. Additionally, the high number of hydrogen bond acceptors suggests that these compounds may face difficulties in membrane transport. According to Ro5, the compounds we designed do not meet the criteria for oral drugs. However, they may be suitable for topical use or intravenous injection, especially when it turns out that they show the expected selectivity of action.
The only compound that partially meets the Ro5 criteria is the potential metabolite 9a. However, our goal was to design selective betulin glycoconjugates capable of releasing biologically active metabolites specifically at the target site, near cancer cells. Therefore, oral administration of the metabolite itself is not a priority in our therapeutic approach.

4. Conclusions

Focusing on a common characteristic of cancer—namely, the increased demand for glucose and the corresponding overexpression of its transporters, as well as the fact that cancer cell growth and their metastasis are strongly related to copper ions uptake—a strategy has been proposed to enhance the efficacy and selectivity of compounds with anticancer activity.
Based on these assumptions, several betulin glycoconjugates were designed and synthesized, which structures and purity were confirmed with spectroscopic analysis. These compounds were subsequently evaluated for cytotoxicity. Even though the determined IC50 values suggest that the described betulin glycoconjugates may not be suitable for use as potential drugs, certain patterns in their structure, influencing the activity and selectivity, have been identified.
The obtained results revealed that the activity of the glycoconjugates is influenced by several factors. These include the type of sugar fragment added, the type of protective groups present in the sugar unit, the absence of protection on the hydroxyl group in the C3 position of betulin, and the length of the linker connecting the sugar components to the betulin derivative. The incorporation of a heteroaromatic 1,2,3-triazole ring, along with the succinyl linker, affects the capacity to chelate metal ions present in many types of cancer cells. The observed improvement in the selectivity of tested glycoconjugates may be because healthy cells are less sensitive to changes in copper ion concentration.
The results presented in this work encourage further optimization of the structure of betulin derivatives. The next logical step appears to be the creation of glycoconjugates that feature an unprotected sugar unit, and checking whether the presence of such a ligand allows for the utilization of their potential against cells that overexpress sugar transporters. Expanding the library of betulin glycoconjugates will facilitate the identification of specific structural elements responsible for eliciting biological activity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers17111737/s1, Details of chemical synthesis and characterization data of compounds, Scheme S1: Synthesis of sugar derivatives 4a–4e, Scheme S2: Synthesis of sugar derivatives 4f–4g, Scheme S3: Synthesis of sugar derivative 4h, NMR spectra of betulin glycoconjugates.

Author Contributions

Conceptualization and methodology, G.P.-G., M.G. and J.S.; synthesis and characterization of chemical compounds, K.W. and J.S.; cytotoxicity tests, J.S.; data curation, M.G., J.S. and K.W.; supervision, G.P.-G. and M.G.; analysis and interpretation of the results, G.P.-G., M.G., K.E. and J.S.; writing—original draft preparation, G.P.-G., M.G. and J.S.; visualization, M.G. and J.S.; writing—review and editing, G.P.-G., M.G. and J.S.; funding acquisition, J.S., M.G. and G.P.-G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported as part of a targeted subsidy for conducting scientific research or development works and related tasks for the development of young scientists and participants of doctoral studies granted by the Ministry of Science and Higher Education, Poland, No. 04/020/BK_25/1113 and by the Rector’s Proquality Grant, Silesian University of Technology (Poland), No. 04/020/RGJ24/1095.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All compounds described in this article are derived from research for a doctoral thesis carried out at the Faculty of Chemistry, Silesian University of Technology. Spectroscopic data of all compounds are available from the authors and will be made available upon sending such a request to the e-mail address: miroslawa.grymel@polsl.pl.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Adepoju, F.O.; Duru, K.C.; Li, E.; Kovaleva, E.G.; Tsurkan, M.V. Pharmacological Potential of Betulin as a Multitarget Compound. Biomolecules 2023, 13, 1105. [Google Scholar] [CrossRef]
  2. Amiri, S.; Dastghaib, S.; Ahmadi, M.; Mehrbod, P.; Khadem, F.; Behrouj, H.; Aghanoori, M.-R.; Machaj, F.; Ghamsari, M.; Rosik, J.; et al. Betulin and Its Derivatives as Novel Compounds with Different Pharmacological Effects. Biotechnol. Adv. 2020, 38, 107409. [Google Scholar] [CrossRef] [PubMed]
  3. Jonnalagadda, S.C.; Suman, P.; Morgan, D.C.; Seay, J.N. Recent Developments on the Synthesis and Applications of Betulin and Betulinic Acid Derivatives as Therapeutic Agents. In Studies in Natural Products Chemistry; Elsevier: Amsterdam, The Netherlands, 2017; Volume 53, pp. 45–84. ISBN 978-0-444-63930-1. [Google Scholar]
  4. Martin, H.; Lázaro, L.R.; Gunnlaugsson, T.; Scanlan, E.M. Glycosidase Activated Prodrugs for Targeted Cancer Therapy. Chem. Soc. Rev. 2022, 51, 9694–9716. [Google Scholar] [CrossRef] [PubMed]
  5. Pastuch-Gawołek, G.; Szreder, J.; Domińska, M.; Pielok, M.; Cichy, P.; Grymel, M. A Small Sugar Molecule with Huge Potential in Targeted Cancer Therapy. Pharmaceutics 2023, 15, 913. [Google Scholar] [CrossRef] [PubMed]
  6. Chen, Z.; Lu, W.; Garcia-Prieto, C.; Huang, P. The Warburg Effect and Its Cancer Therapeutic Implications. J. Bioenerg. Biomembr. 2007, 39, 267–274. [Google Scholar] [CrossRef]
  7. Liberti, M.V.; Locasale, J.W. The Warburg Effect: How Does It Benefit Cancer Cells? Trends Biochem. Sci. 2016, 41, 211–218. [Google Scholar] [CrossRef]
  8. Pliszka, M.; Szablewski, L. Glucose Transporters as a Target for Anticancer Therapy. Cancers 2021, 13, 4184. [Google Scholar] [CrossRef]
  9. Ancey, P.; Contat, C.; Meylan, E. Glucose Transporters in Cancer—From Tumor Cells to the Tumor Microenvironment. FEBS J. 2018, 285, 2926–2943. [Google Scholar] [CrossRef] [PubMed]
  10. Calvo, M.B.; Figueroa, A.; Pulido, E.G.; Campelo, R.G.; Aparicio, L.A. Potential Role of Sugar Transporters in Cancer and Their Relationship with Anticancer Therapy. Int. J. Endocrinol. 2010, 2010, 205357. [Google Scholar] [CrossRef]
  11. Sakashita, M.; Aoyama, N.; Minami, R.; Maekawa, S.; Kuroda, K.; Shirasaka, D.; Ichihara, T.; Kuroda, Y.; Maeda, S.; Kasuga, M. Glut1 Expression in T1 and T2 Stage Colorectal Carcinomas. Eur. J. Cancer 2001, 37, 204–209. [Google Scholar] [CrossRef]
  12. Grover-McKay, M.; Walsh, S.A.; Seftor, E.A.; Thomas, P.A.; Hendrix, M.J. Role for Glucose Transporter 1 Protein in Human Breast Cancer. Pathol. Oncol. Res. 1998, 4, 115–120. [Google Scholar] [CrossRef]
  13. Wu, M.; Neilson, A.; Swift, A.L.; Moran, R.; Tamagnine, J.; Parslow, D.; Armistead, S.; Lemire, K.; Orrell, J.; Teich, J.; et al. Multiparameter Metabolic Analysis Reveals a Close Link between Attenuated Mitochondrial Bioenergetic Function and Enhanced Glycolysis Dependency in Human Tumor Cells. Am. J. Physiol. Cell Physiol. 2007, 292, C125–C136. [Google Scholar] [CrossRef] [PubMed]
  14. Lai, J.-H.; Jan, H.-J.; Liu, L.-W.; Lee, C.-C.; Wang, S.-G.; Hueng, D.-Y.; Cheng, Y.-Y.; Lee, H.-M.; Ma, H.-I. Nodal Regulates Energy Metabolism in Glioma Cells by Inducing Expression of Hypoxia-Inducible Factor 1. Neuro-Oncol. 2013, 15, 1330–1341. [Google Scholar] [CrossRef] [PubMed]
  15. Lacombe, D. Glufosfamide: Can We Improve the Process of Anticancer Agent Development? Expert Opin. Investig. Drugs 2012, 21, 749–754. [Google Scholar] [CrossRef]
  16. Porter, J.; Noble, A.R.; Signoret, N.; Fascione, M.A.; Miller, G.J. Exploring a Gemcitabine-Glucose Hybrid as a Glycoconjugate Prodrug. ACS Omega 2024, 9, 31703–31713. [Google Scholar] [CrossRef]
  17. Vaidya, S.P.; Patra, M. Platinum Glycoconjugates: “Sweet Bullets” for Targeted Cancer Therapy? Curr. Opin. Chem. Biol. 2023, 72, 102236. [Google Scholar] [CrossRef]
  18. Iorio, A.L.; Lenci, E.; Marzano, C.; Bucaletti, E.; Tirinnanzi, B.; Casati, G.; Giunti, L.; Dallari, C.; Credi, C.; Sardi, I.; et al. Oxime Linked Doxorubicin Glycoconjugates Improve the Specific Targeting of Glioblastoma in High-Grade Glioma Therapy. ACS Med. Chem. Lett. 2024, 15, 1953–1960. [Google Scholar] [CrossRef]
  19. Meng, X.; Lian, X.; Li, X.; Ya, Q.; Li, T.; Zhang, Y.; Yang, Y.; Zhang, Y. Synthesis of 2′-Paclitaxel 2-Deoxy-2-Fluoro-Glucopyranosyl Carbonate for Specific Targeted Delivery to Cancer Cells. Carbohydr. Res. 2020, 493, 108034. [Google Scholar] [CrossRef] [PubMed]
  20. Vitaku, E.; Smith, D.T.; Njardarson, J.T. Analysis of the Structural Diversity, Substitution Patterns, and Frequency of Nitrogen Heterocycles among U.S. FDA Approved Pharmaceuticals: Miniperspective. J. Med. Chem. 2014, 57, 10257–10274. [Google Scholar] [CrossRef]
  21. Kumar, D.; Kumar Jain, S. A Comprehensive Review of N-Heterocycles as Cytotoxic Agents. Curr. Med. Chem. 2016, 23, 4338–4394. [Google Scholar] [CrossRef]
  22. Xu, Z.; Zhao, S.-J.; Liu, Y. 1,2,3-Triazole-Containing Hybrids as Potential Anticancer Agents: Current Developments, Action Mechanisms and Structure-Activity Relationships. Eur. J. Med. Chem. 2019, 183, 111700. [Google Scholar] [CrossRef] [PubMed]
  23. Dheer, D.; Singh, V.; Shankar, R. Medicinal Attributes of 1,2,3-Triazoles: Current Developments. Bioorg. Chem. 2017, 71, 30–54. [Google Scholar] [CrossRef] [PubMed]
  24. Da S. M. Forezi, L.; Lima, C.G.S.; Amaral, A.A.P.; Ferreira, P.G.; De Souza, M.C.B.V.; Cunha, A.C.; De C. Da Silva, F.; Ferreira, V.F. Bioactive 1,2,3-Triazoles: An Account on Their Synthesis, Structural Diversity and Biological Applications. Chem. Rec. 2021, 21, 2782–2807. [Google Scholar] [CrossRef]
  25. Markham, A. Savolitinib: First Approval. Drugs 2021, 81, 1665–1670. [Google Scholar] [CrossRef] [PubMed]
  26. Yamansarov, E.Y.; Lopatukhina, E.V.; Evteev, S.A.; Skvortsov, D.A.; Lopukhov, A.V.; Kovalev, S.V.; Vaneev, A.N.; Shkil’, D.O.; Akasov, R.A.; Lobov, A.N.; et al. Discovery of Bivalent GalNAc-Conjugated Betulin as a Potent ASGPR-Directed Agent against Hepatocellular Carcinoma. Bioconjugate Chem. 2021, 32, 763–781. [Google Scholar] [CrossRef]
  27. Bori, I.D.; Hung, H.-Y.; Qian, K.; Chen, C.-H.; Morris-Natschke, S.L.; Lee, K.-H. Anti-AIDS Agents 88. Anti-HIV Conjugates of Betulin and Betulinic Acid with AZT Prepared via Click Chemistry. Tetrahedron Lett. 2012, 53, 1987–1989. [Google Scholar] [CrossRef]
  28. Grymel, M.; Pastuch-Gawołek, G.; Lalik, A.; Zawojak, M.; Boczek, S.; Krawczyk, M.; Erfurt, K. Glycoconjugation of Betulin Derivatives Using Copper-Catalyzed 1,3-Dipolar Azido-Alkyne Cycloaddition Reaction and a Preliminary Assay of Cytotoxicity of the Obtained Compounds. Molecules 2020, 25, 6019. [Google Scholar] [CrossRef]
  29. Bębenek, E.; Jastrzębska, M.; Kadela-Tomanek, M.; Chrobak, E.; Orzechowska, B.; Zwolińska, K.; Latocha, M.; Mertas, A.; Czuba, Z.; Boryczka, S. Novel Triazole Hybrids of Betulin: Synthesis and Biological Activity Profile. Molecules 2017, 22, 1876. [Google Scholar] [CrossRef]
  30. Shi, W.; Tang, N.; Yan, W.-D. Synthesis and Cytotoxicity of Triterpenoids Derived from Betulin and Betulinic Acid via Click Chemistry. J. Asian Nat. Prod. Res. 2015, 17, 159–169. [Google Scholar] [CrossRef]
  31. Antimonova, A.N.; Petrenko, N.I.; Shakirov, M.M.; Rybalova, T.V.; Frolova, T.S.; Shul’ts, E.E.; Kukina, T.P.; Sinitsyna, O.I.; Tolstikov, G.A. Synthesis and Study of Mutagenic Properties of Lupane Triterpenoids Containing 1,2,3-Triazole Fragments in the C-30 Position. Chem. Nat. Compd. 2013, 49, 657–664. [Google Scholar] [CrossRef]
  32. Bębenek, E.; Kadela-Tomanek, M.; Chrobak, E.; Jastrzębska, M.; Książek, M. Synthesis and Structural Characterization of a New 1,2,3-Triazole Derivative of Pentacyclic Triterpene. Crystals 2022, 12, 422. [Google Scholar] [CrossRef]
  33. Khalil, M.M.; Radalla, A.M.; Abd Elnaby, N.M. Solution Equilibria and Thermodynamic Studies of Complexation of Divalent Transition Metal Ions with Some Triazoles and Biologically Important Aliphatic Dicarboxylic Acids in Aqueous Media. J. Solut. Chem. 2013, 42, 1123–1145. [Google Scholar] [CrossRef]
  34. Chen, L.; Min, J.; Wang, F. Copper Homeostasis and Cuproptosis in Health and Disease. Signal Transduct. Target. Ther. 2022, 7, 378. [Google Scholar] [CrossRef] [PubMed]
  35. Santini, C.; Pellei, M.; Gandin, V.; Porchia, M.; Tisato, F.; Marzano, C. Advances in Copper Complexes as Anticancer Agents. Chem. Rev. 2014, 114, 815–862. [Google Scholar] [CrossRef]
  36. Gupte, A.; Mumper, R.J. Elevated Copper and Oxidative Stress in Cancer Cells as a Target for Cancer Treatent. Cancer Treat. Rev. 2009, 35, 32–46. [Google Scholar] [CrossRef]
  37. Denoyer, D.; Masaldan, S.; La Fontaine, S.; Cater, M.A. Targeting Copper in Cancer Therapy: ‘Copper That Cancer’. Metallomics 2015, 7, 1459–1476. [Google Scholar] [CrossRef]
  38. Thibeault, D.; Gauthier, C.; Legault, J.; Bouchard, J.; Dufour, P.; Pichette, A. Synthesis and Structure–Activity Relationship Study of Cytotoxic Germanicane- and Lupane-Type 3β-O-Monodesmosidic Saponins Starting from Betulin. Bioorg. Med. Chem. 2007, 15, 6144–6157. [Google Scholar] [CrossRef]
  39. Grymel, M.; Lalik, A.; Kazek-Kęsik, A.; Szewczyk, M.; Grabiec, P.; Erfurt, K. Design, Synthesis and Preliminary Evaluation of the Cytotoxicity and Antibacterial Activity of Novel Triphenylphosphonium Derivatives of Betulin. Molecules 2022, 27, 5156. [Google Scholar] [CrossRef]
  40. Domińska, M.; Pastuch-Gawołek, G.; Skonieczna, M.; Szeja, W.; Domiński, A.; Kurcok, P. Glycoconjugation of Quinoline Derivatives Using the C-6 Position in Sugars as a Strategy for Improving the Selectivity and Cytotoxicity of Functionalized Compounds. Molecules 2022, 27, 6918. [Google Scholar] [CrossRef]
  41. Le Roux, A.; Meunier, S.; Le Gall, T.; Denis, J.; Bischoff, P.; Wagner, A. Synthesis and Radioprotective Properties of Pulvinic Acid Derivatives. ChemMedChem 2011, 6, 561–569. [Google Scholar] [CrossRef]
  42. Ibatullin, F.M.; Shabalin, K.A. A Simple and Convenient Synthesis of Glycosyl Azides. Synth. Commun. 2000, 30, 2819–2823. [Google Scholar] [CrossRef]
  43. Brzuska, G.; Pastuch-Gawolek, G.; Krawczyk, M.; Szewczyk, B.; Krol, E. Anti-Tick-Borne Encephalitis Virus Activity of Novel Uridine Glycoconjugates Containing Amide or/and 1,2,3-Triazole Moiety in the Linker Structure. Pharmaceuticals 2020, 13, 460. [Google Scholar] [CrossRef]
  44. Pastuch-Gawołek, G.; Malarz, K.; Mrozek-Wilczkiewicz, A.; Musioł, M.; Serda, M.; Czaplinska, B.; Musiol, R. Small Molecule Glycoconjugates with Anticancer Activity. Eur. J. Med. Chem. 2016, 112, 130–144. [Google Scholar] [CrossRef]
  45. Joosten, J.A.F.; Loimaranta, V.; Appeldoorn, C.C.M.; Haataja, S.; El Maate, F.A.; Liskamp, R.M.J.; Finne, J.; Pieters, R.J. Inhibition of Streptococcus s Uis Adhesion by Dendritic Galabiose Compounds at Low Nanomolar Concentration. J. Med. Chem. 2004, 47, 6499–6508. [Google Scholar] [CrossRef]
  46. Calvaresi, E.C.; Hergenrother, P.J. Glucose Conjugation for the Specific Targeting and Treatment of Cancer. Chem. Sci. 2013, 4, 2319. [Google Scholar] [CrossRef] [PubMed]
  47. Fu, J.; Yang, J.; Seeberger, P.H.; Yin, J. Glycoconjugates for Glucose Transporter-Mediated Cancer-Specific Targeting and Treatment. Carbohydr. Res. 2020, 498, 108195. [Google Scholar] [CrossRef] [PubMed]
  48. Hossain, F.; Andreana, P.R. Developments in Carbohydrate-Based Cancer Therapeutics. Pharmaceuticals 2019, 12, 84. [Google Scholar] [CrossRef]
  49. Barnett, J.E.G.; Holman, G.D.; Munday, K.A. Structural Requirements for Binding to the Sugar-Transport System of the Human Erythrocyte. Biochem. J. 1973, 131, 211–221. [Google Scholar] [CrossRef]
  50. Patra, M.; Johnstone, T.C.; Suntharalingam, K.; Lippard, S.J. A Potent Glucose–Platinum Conjugate Exploits Glucose Transporters and Preferentially Accumulates in Cancer Cells. Angew. Chem. Int. Ed. 2016, 55, 2550–2554. [Google Scholar] [CrossRef]
  51. Tanasova, M.; Fedie, J.R. Molecular Tools for Facilitative Carbohydrate Transporters (Gluts). ChemBioChem 2017, 18, 1774–1788. [Google Scholar] [CrossRef]
  52. Deng, D.; Xu, C.; Sun, P.; Wu, J.; Yan, C.; Hu, M.; Yan, N. Crystal Structure of the Human Glucose Transporter GLUT1. Nature 2014, 510, 121–125. [Google Scholar] [CrossRef] [PubMed]
  53. Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef] [PubMed]
  54. Yang, J.; Wen, J.; Tian, T.; Lu, Z.; Wang, Y.; Wang, Z.; Wang, X.; Yang, Y. GLUT-1 Overexpression as an Unfavorable Prognostic Biomarker in Patients with Colorectal Cancer. Oncotarget 2017, 8, 11788–11796. [Google Scholar] [CrossRef]
  55. Shin, E.; Koo, J.S. Glucose Metabolism and Glucose Transporters in Breast Cancer. Front. Cell Dev. Biol. 2021, 9, 728759. [Google Scholar] [CrossRef] [PubMed]
  56. Laudański, P.; Swiatecka, J.; Kovalchuk, O.; Wołczyński, S. Expression of GLUT1 Gene in Breast Cancer Cell Lines MCF-7 and MDA-MB-231. Ginekol. Pol. 2003, 74, 782–785. [Google Scholar]
  57. O’Connor, P.M.; Jackman, J.; Bae, I.; Myers, T.G.; Fan, S.; Mutoh, M.; Scudiero, D.A.; Monks, A.; Sausville, E.A.; Weinstein, J.N.; et al. Characterization of the P53 Tumor Suppressor Pathway in Cell Lines of the National Cancer Institute Anticancer Drug Screen and Correlations with the Growth-Inhibitory Potency of 123 Anticancer Agents. Cancer Res. 1997, 57, 4285–4300. [Google Scholar]
  58. Koygun, G.; Arslan, E.; Zengin, G.; Orlando, G.; Ferrante, C. Comparison of Anticancer Activity of Dorycnium Pentaphyllum Extract on MCF-7 and MCF-12A Cell Line: Correlation with Invasion and Adhesion. Biomolecules 2021, 11, 671. [Google Scholar] [CrossRef]
  59. Larsson, S.; Rydén, T.; Holst, U.; Oredsson, S.; Johansson, M. Estimating the Total Rate of DNA Replication Using Branching Processes. Bull. Math. Biol. 2008, 70, 2177–2194. [Google Scholar] [CrossRef]
  60. Feng, L.; Dong, Z.; Tao, D.; Zhang, Y.; Liu, Z. The Acidic Tumor Microenvironment: A Target for Smart Cancer Nano-Theranostics. Natl. Sci. Rev. 2018, 5, 269–286. [Google Scholar] [CrossRef]
  61. Kato, Y.; Ozawa, S.; Miyamoto, C.; Maehata, Y.; Suzuki, A.; Maeda, T.; Baba, Y. Acidic Extracellular Microenvironment and Cancer. Cancer Cell. Int. 2013, 13, 89. [Google Scholar] [CrossRef]
  62. Hu, K.; Guo, J.; Zeng, J.; Shao, Y.; Wu, B.; Mo, J.; Mo, G. Current State of Research on Copper Complexes in the Treatment of Breast Cancer. Open Life Sci. 2024, 19, 20220840. [Google Scholar] [CrossRef] [PubMed]
  63. Guan, D.; Zhao, L.; Shi, X.; Ma, X.; Chen, Z. Copper in Cancer: From Pathogenesis to Therapy. Biomed. Pharmacother. 2023, 163, 114791. [Google Scholar] [CrossRef] [PubMed]
  64. Barbosa, A.M.; Martel, F. Targeting Glucose Transporters for Breast Cancer Therapy: The Effect of Natural and Synthetic Compounds. Cancers 2020, 12, 154. [Google Scholar] [CrossRef]
  65. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings. Adv. Drug Deliv. Rev. 2001, 46, 3–26. [Google Scholar] [CrossRef] [PubMed]
  66. Doak, B.C.; Over, B.; Giordanetto, F.; Kihlberg, J. Oral Druggable Space beyond the Rule of 5: Insights from Drugs and Clinical Candidates. Chem. Biol. 2014, 21, 1115–1142. [Google Scholar] [CrossRef]
  67. Price, E.; Weinheimer, M.; Rivkin, A.; Jenkins, G.; Nijsen, M.; Cox, P.B.; DeGoey, D. Beyond Rule of Five and PROTACs in Modern Drug Discovery: Polarity Reducers, Chameleonicity, and the Evolving Physicochemical Landscape. J. Med. Chem. 2024, 67, 5683–5698. [Google Scholar] [CrossRef]
Cancers 17 01737 g001
Figure 1. Chemical structure of betulin with indication of items for possible derivatization.
Figure 1. Chemical structure of betulin with indication of items for possible derivatization.
Cancers 17 01737 g001
Cancers 17 01737 g002
Figure 2. Selected structural modifications of betulin skeleton at positions C3, C28, and C30 via CuAAC method [26,27,28,29,30,31,32].
Figure 2. Selected structural modifications of betulin skeleton at positions C3, C28, and C30 via CuAAC method [26,27,28,29,30,31,32].
Cancers 17 01737 g002
Cancers 17 01737 g003
Figure 3. Betulin glycoconjugates with a 1,2,3-triazole linker at the C28 position or the C3 and C28 positions [28].
Figure 3. Betulin glycoconjugates with a 1,2,3-triazole linker at the C28 position or the C3 and C28 positions [28].
Cancers 17 01737 g003
Cancers 17 01737 g004
Figure 4. Structures of a new type of BN glycoconjugates.
Figure 4. Structures of a new type of BN glycoconjugates.
Cancers 17 01737 g004
Cancers 17 01737 sch001
Scheme 1. General scheme of two possible approaches to glycoconjugates synthesis with click chemistry concept. (A) Reaction of propargyl derivative of betulin with sugar azide, (B) Reaction of an azide derivative of betulin with a propargyl sugar derivative.
Scheme 1. General scheme of two possible approaches to glycoconjugates synthesis with click chemistry concept. (A) Reaction of propargyl derivative of betulin with sugar azide, (B) Reaction of an azide derivative of betulin with a propargyl sugar derivative.
Cancers 17 01737 sch001
Cancers 17 01737 sch002
Scheme 2. Synthesis of BN glycoconjugates (5–8) and potential metabolite (9a). Reagents and Conditions: (a) propargyl bromide, K2CO3, DMF, r.t., 5 days; (b) CuSO4·5H2O, NaAsc, i-PrOH/THF/H2O (1:1:1, v:v:v), r.t., 7 days.
Scheme 2. Synthesis of BN glycoconjugates (5–8) and potential metabolite (9a). Reagents and Conditions: (a) propargyl bromide, K2CO3, DMF, r.t., 5 days; (b) CuSO4·5H2O, NaAsc, i-PrOH/THF/H2O (1:1:1, v:v:v), r.t., 7 days.
Cancers 17 01737 sch002
Cancers 17 01737 g005
Figure 5. Structures of sugar derivatives used for glycoconjugation of betulin.
Figure 5. Structures of sugar derivatives used for glycoconjugation of betulin.
Cancers 17 01737 g005
Cancers 17 01737 g006
Figure 6. Dependence of HCT 116 and MCF-7 cells’ proliferation on concentration of BN glycoconjugates as estimated using MTT assay compared to BN alone: (A) proliferation of HCT 116 cells after 24 h incubation, (B) proliferation of HCT 116 cells after 48 h incubation, (C) proliferation of HCT 116 cells after 48 h incubation in presence of 20 µM copper ions (Cu2+), (D) proliferation of MCF-7 cells after 72 h incubation, (E) proliferation of MCF-7 cells after 72 h incubation in presence of 20 µM copper ions (Cu2+). Data are presented as mean ± standard deviation (n = 3). Statistical analysis was performed two-way ANOVA with replication followed by post hoc t-tests with Bonferroni correction. (*) p-value < 0.05, in comparison to untreated control.
Figure 6. Dependence of HCT 116 and MCF-7 cells’ proliferation on concentration of BN glycoconjugates as estimated using MTT assay compared to BN alone: (A) proliferation of HCT 116 cells after 24 h incubation, (B) proliferation of HCT 116 cells after 48 h incubation, (C) proliferation of HCT 116 cells after 48 h incubation in presence of 20 µM copper ions (Cu2+), (D) proliferation of MCF-7 cells after 72 h incubation, (E) proliferation of MCF-7 cells after 72 h incubation in presence of 20 µM copper ions (Cu2+). Data are presented as mean ± standard deviation (n = 3). Statistical analysis was performed two-way ANOVA with replication followed by post hoc t-tests with Bonferroni correction. (*) p-value < 0.05, in comparison to untreated control.
Cancers 17 01737 g006
Cancers 17 01737 g007
Figure 7. Dependence of NHDF-Neo cells’ proliferation on concentration of BN glycoconjugates, as estimated using MTT assay compared to BN alone, in absence (A) and presence (B) of 20 µM of Cu2+ after 24 h. Data are presented as mean ± standard deviation (n = 3). Statistical significance was calculated from two-way ANOVA with replication, followed by post hoc t-tests with Bonferroni correction, where p < 0.05 and is indicated by an asterisk (*).
Figure 7. Dependence of NHDF-Neo cells’ proliferation on concentration of BN glycoconjugates, as estimated using MTT assay compared to BN alone, in absence (A) and presence (B) of 20 µM of Cu2+ after 24 h. Data are presented as mean ± standard deviation (n = 3). Statistical significance was calculated from two-way ANOVA with replication, followed by post hoc t-tests with Bonferroni correction, where p < 0.05 and is indicated by an asterisk (*).
Cancers 17 01737 g007
Table 1. Yields and HRMS date of obtained betulin glycoconjugates (5a–5c, 6a, 6b, 7a–7d, 8a) and potential metabolite 9a.
Table 1. Yields and HRMS date of obtained betulin glycoconjugates (5a–5c, 6a, 6b, 7a–7d, 8a) and potential metabolite 9a.
BN AnalogueSugarProductHRMS ([M+Na]+)
No.R1R2Yield (%)Summary FormulaCalc.Found.
3a4a5aOAcOAc72C53H75N3O151018.52521018.5353
3a4b5bOAcOBn60C73H93N3O111210.67081210.7081
3b4a5cOHOAc64C51H75N3O14976.5147976.5180
3a4c6aOAcOAc71C53H75N3O151018.52521018.5360
3a4d6bOAcOBn53C73H93N3O111210.67081210.6998
3a4f7aOAcOAc73C55H81N3O161062.55151062.5526
3a4g7bOAcOAc69C56H83N3O161076.56711076.5715
3b4f7cOHOAc59C53H79N3O151020.54091020.5486
3b4g7dOHOAc70C54H81N3O151034.55651034.5626
3a4h8aOAcOAc12C53H75N3O151018.52521018.5356
3a2-azidoethanol9aOAc-32C41H63N3O7732.4564732.4573
Table 2. Cytotoxic activity of betulin (BN), betulin glycoconjugates (5a–5c, 6a, 6b, 7a–7d, 8a), and metabolite 9a a.
Table 2. Cytotoxic activity of betulin (BN), betulin glycoconjugates (5a–5c, 6a, 6b, 7a–7d, 8a), and metabolite 9a a.
CompoundHCT 116MCF-7NHDF-Neo
IC50 [µM] bIC50 [µM] cSIIC50 [µM] dSIIC50 [µM] b
BN34.88 ± 4.2625.96 ± 5.401.317.89 ± 1.061.934.90 ± 8.36
BN + Cu2+ e-20.76 ± 2.711.613.91 ± 2.352.433.34 ± 5.97
5a>200>200-163.12 ± 7.65>1.2>200
5a + Cu2+ e->200-108.51 ± 5.85>1.8>200
5b>200>200-167.61 ± 11.66>1.2>200
5c>200>200-128.07 ± 1.35>1.6>200
5c + Cu2+ e->200-70.71 ± 5.44>2.8>200
6a>200>200-122.61 ± 6.84>1.6>200
6a + Cu2+ e->200-59.81 ± 1.85>3.3>200
6b>200>200-184.13 ± 7.59>1.1>200
7a>200>200--144.12 ± 4.70>1.4>200
7a + Cu2+ e-150.08 ± 8.01>1.3135.01 ± 10.23>1.5>200
7b>200>200 152.23 ± 2.91>1.3>200
7b + Cu2+ e-112.48 ± 0.57>1.8124.29 ± 1.64>1.6>200
7c>200>200-134.92 ± 7.92>1.5>200
7c + Cu2+ e->200-117.94 ± 1.42>1.7>200
7d>200131.16 ± 8.76>1.5165.02 ± 8.32>1.2>200
7d + Cu2+ e-60.40 ± 6.95>3.3147.02 ± 2.32>1.4>200
8a>200>200-152.63 ± 1.98>1.3>200
8a + Cu2+ e->200-88.33 ± 3.31>2.3>200
9a>200>200 147.56 ± 8.71>1.4>200
9a + Cu2+ e>200>200 138.8 ± 6.21>1,4>200
Doxorubicin13.17 ± 0.58 1.89 ± 0.19>10.60.29 ± 0.04>70>20
a Cytotoxicity was assessed by MTT assay; incubation time: b 24 h; c 48 h; d 72 h, e Addition of CuCl2 at 20 µM. SI: selectivity index; ratio of IC50 of healthy cells (NHDF-Neo) to IC50 of cancer cells.
Table 3. Molecular descriptors analysis of BN glycoconjugates using Molinspiration 2025 online software tools (http://www.molinspiration.com; accessed on 8 February 2025).
Table 3. Molecular descriptors analysis of BN glycoconjugates using Molinspiration 2025 online software tools (http://www.molinspiration.com; accessed on 8 February 2025).
StructurepLogPTPSA
(Å2)		MW
(g/mol)		Vol
(Å3)		nONnOHNHnrotb
5a7.96224.09996.21930.3618022
5b9.94155.801188.561141.0314026
5c7.25218.01954.17893.8517120
6a7.96224.09996.21930.3618022
6b9.94155.801188.561141.0314026
7a7.94233.321040.26972.9519025
7b8.19233.321054.29989.7519026
7c7.24227.24998.22936.4418123
7d7.51227.241012.25953.2418124
8a7.96224.09996.21930.3618022
9a7.27129.86709.97694.7810114
pLogP: predicted LogP; TPSA: topological polar surface area; MW: molecular weight; Vol: molecular volume; nON: number of hydrogen bond acceptors; nOHNH: number of hydrogen bond donors; nrotb: number of rotatable bonds.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Szreder, J.; Woźniak, K.; Erfurt, K.; Grymel, M.; Pastuch-Gawołek, G. Synthesis and Preliminary Cytotoxicity Evaluation of 3-Lup-20(29)-Ene-3β,28-Diol Glycoconjugates Containing a Succinic Linker and a 1,2,3-Triazole Ring. Cancers 2025, 17, 1737. https://doi.org/10.3390/cancers17111737

AMA Style

Szreder J, Woźniak K, Erfurt K, Grymel M, Pastuch-Gawołek G. Synthesis and Preliminary Cytotoxicity Evaluation of 3-Lup-20(29)-Ene-3β,28-Diol Glycoconjugates Containing a Succinic Linker and a 1,2,3-Triazole Ring. Cancers. 2025; 17(11):1737. https://doi.org/10.3390/cancers17111737

Chicago/Turabian Style

Szreder, Julia, Klaudia Woźniak, Karol Erfurt, Mirosława Grymel, and Gabriela Pastuch-Gawołek. 2025. "Synthesis and Preliminary Cytotoxicity Evaluation of 3-Lup-20(29)-Ene-3β,28-Diol Glycoconjugates Containing a Succinic Linker and a 1,2,3-Triazole Ring" Cancers 17, no. 11: 1737. https://doi.org/10.3390/cancers17111737

APA Style

Szreder, J., Woźniak, K., Erfurt, K., Grymel, M., & Pastuch-Gawołek, G. (2025). Synthesis and Preliminary Cytotoxicity Evaluation of 3-Lup-20(29)-Ene-3β,28-Diol Glycoconjugates Containing a Succinic Linker and a 1,2,3-Triazole Ring. Cancers, 17(11), 1737. https://doi.org/10.3390/cancers17111737

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop